The ability to efficiently incorporate shRNA expression cassettes into viral vector gene delivery systems significantly expands the spectrum of research and clinical applications of the shRNA technology. HIV-1 and Adeno Associated Virus (AAV) based vectors are two of the leading vectors in the arena of in vivo and in vitro gene delivery. AAV based vectors are highly efficient at gene delivery in vivo, specifically in non-dividing cells such as liver, heart, muscle, and brain with long-term gene expression (up to 6 yr. in monkey and now 3.7 yr in human). These non-integrating vectors have a packaging capacity of 5 kb and have not been the vector of choice when targeting dividing cells such as stem cells, cancer cells, and rapidly growing cells in vivo. On the other hand, the integrating HIV-1 based vectors efficiently transduce and maintain transgene expression in various stem cells including early embryos, and hematopoietic stem cells. Low vector titers, limitations in in vivo gene delivery, and biosafety concerns are noted weaknesses of the newer HIV-1 vector system. Taken together, these two robust viral gene delivery systems complement each other and provide ability to transduce most target organs for both research and clinical applications. In response to the RFA- HL-05-019, "The purpose of this RFA is to stimulate research towards (1) understanding ..., (2) assessing..., and (3) determining optimal delivery methods for uptake by the target tissues." and in the spirit of a R21, we propose to optimize two well established viral vector delivery systems (Lenti & AAV) for shRNA delivery and provide these reagents to the research community for utilization in both in vitro and in vivo shRNA delivery. The primary objective of this R21 is to generate novel AAV and Lentiviral vectors for the research community to exploit efficient delivery of shRNA cassettes both in vitro and in vivo. Specifically, we propose to develop cell type/receptor-specific AAV mutants obtained through directed evolution into scAAV vectors for gene delivery in vivo. Novel AAV mutants that selectively (a) transduce primary cell lines including airway, hepatocytes, or skeletal muscle cells; (b) traverse barrier epithelial/endothelial cells; or (c) transduce target tissue in vivo will be isolated and characterized for shRNA delivery. In addition, we will develop a universal third generation stable HIV-1 vector producer cell line, which will facilitate the production of large amounts of high titer self-inactivating (SIN) HIV-1 vectors carrying shRNA expression cassettes. To this end we will employ the FLIP recombinase system as a means to incorporate shRNA expression cassettes into an HIV- 1 vector integrated in a third generation high titer producer cell line. This approach, which facilitates scaling up safe HIV-1 vector production, increases the likelihood of employing shRNA-expressing HIV-1 vectors in clinical trials. The long-term objective is to improve on existing AAV and HIV vectors and provide novel delivery reagents for shRNA expression cassettes. [unreadable] [unreadable] [unreadable]